Method for electronically detecting at least one specific interaction between probe molecules and target biomolecules

ABSTRACT

The invention concerns a method for detecting at least one specific interaction between probe molecules and target biomiolecules fixed to at least one active zone of a sensor. Said sensor consists of an array of field-effect transistors (T 1 , T 2 ,), each of which has a gate region constituting an active zone ( 3 ) whereon said specific interaction is to be detected.

The present invention relates to a method for electronically detectingat least one interaction between molecules and target biomolecules.

A method for detecting the hybridization of DNA sequences using afield-effect transistor is already known, as was described in thearticle by E. Souteyrand et al., entitled “Direct detection of thehybridization of synthetic homo-oligomer DNA sequences by field effect”,published in 1997 in J. Phys. Chem. B1997, 101, pages 2980 to 2985. Atransistor of the ISFET (“ion-sensitive field-effect transistor”) typewhich can be used in this type of application has been described in thearticle by Piet Bergveld “Development, operation and application of theISFET as a tool for electrophysiology”, published in IEEE Transactionson Biomedical Engineering, volume BME-19—No. 5, September 1972, pages342 to 351. Indications on the manufacture of such transistor structurescan be found in the article by V. Kiessling et al., entitled“Extracellular resistance in cell adhesion measured with a transistorprobe”, published in Langmuir 2000, 16, pages 3517-3521. Finally, asurface preparation method has been described in the article by A. Kumaret al., entitled “Silanized nucleic acid: a general platform for DNAimmobilization”, published in Nucleic Acid Research 2000, volume 28, No.14, pages i to vi.

Two methods for fixing molecular probes to the surface can in particularbe used in the context of the present invention. The first consists ofdirect synthesis on a solid phase, as described, for example, in thearticle by S. P. A. Fodor et al., entitled “Light-directed, spatiallyaddressable parallel chemical synthesis”, published in Science 251,pages 767 to 773 (1991). The second is the fixing of the molecules usinga dilution.

In order to obtain a correct detection of the interactions betweenbiomolecules, it is advisable to take into account a certain amount ofparameters:

A—Sensitivity

It can be very good (comparable to that of detection by fluorescence),but it depends on the condition of the molecular layers on the activesurfaces. For the transistor-type sensors which are used here, therequired amount of biological molecules (probes and targets) isinversely proportional to the surface area of the sensor. It istherefore advantageous to miniaturize the structures, but attention mustbe paid to the signal/noise ratio.

B—Dynamics

The range of the various concentrations of molecule that can be measuredis narrow. At low concentration, it is possible to be limited byparasitic signals (for example, it is sometimes possible to already seea signal induced by a drop of pure water that had dried on the surface).At high concentration, saturation is observed when the effective chargeapproaches zero.

C—Specificity

The term “specificity” is intended to mean the ability of the system todistinguish between two types of different target molecules. Forhybridization between DNA molecules, for example, this difference may bea difference in the sequence of the base pairs. It is a question ofoptimizing the conditions (salt, temperature, duration and, optionally,pH) of the intermolecular recognition reaction such that the specificinteraction (for example, the hybridization between complementarysequences) dominates with respect to nonspecific processes (for example,adsorption, nonspecific, ionic and hydrophobic interactions). Theseoptimized conditions do not generally correspond to the best conditionsfor an electronic detection. For example, more salt is typically usedfor a specific hybridization than for an electronic detection. Acompromise also has to be found for the distance of the probe moleculeswith respect to the surface. A small distance is favorable for theelectronic detection because of electrostatic screening, but is oftenunfavorable for the specificity. If the probe molecules are too close tothe SiO₂ surface, the nonspecific molecule/surface interactions canbecome dominant. In addition, the specific interaction between the probeand target biomolecules can be hindered by a surface that is in thevicinity (problem of accessibility or steric hindrance).

These effects depend on the molecular layers deposited on the activesurface of the transistor array (and in particular on their chargestate).

In the description hereinafter, an experimental approach that makes itpossible to achieve the desired result has been proposed. This approachhas made it possible to demonstrate a key point of the detection, whichconstitutes the basic idea of the present invention, namely that it wasadvisable to separate the recognition reaction from the detection step.

The present invention thus relates to a method for electronicallydetecting at least one specific interaction between probe moleculesfixed to at least one active zone of a sensor and target biomolecules,characterized in that said sensor consists of an array of field-effecttransistors (T₁, T₂, etc.), each of which has a source region (5), adrain region (D), and a gate region which constitutes an active zone (3)on which said specific interaction is to be detected, and in that itcomprises the following steps:

a) bringing at least one active zone (3) into contact with probemolecules of a given type fixed to said active zone,

b) bringing at least some of the probe molecules into contact withtarget biomolecules capable of interaction with said probe molecules,and performing a said interaction in a reaction buffer having a firstsalt concentration,

c) measuring at least one point of the drain current/source-gatevoltage/source-drain voltage characteristic of at least one transistorof said array to detect said specific interaction at least for ameasurement point obtained in a measuring buffer having a second saltconcentration that is lower than the first concentration for probemolecules having been subjected to said specific interaction. Step c canbe carried out differentially between said measurement point and areference point, in particular in a said measuring buffer, for probemolecules of the same type that have not been subjected to said specificinteraction.

In general, a probe molecule is considered to be any molecule, inparticular biomolecules (DNA, RNA, proteins, etc), or else chemicallysynthesized oligonucleotide DNA, or alternatively peptide nucleic acids(PNAs), which can be grafted onto said active surface and which can bespecifically recognized by a target biomolecule.

According to a “temporal” variant, the differential measurement of stepc) is carried out on the same probe molecules, before the interaction ofstep b).

According to a first “spatial” variant, the differential measurement ofstep c) is carried out between two groups of probe molecules of the sametype, for example (DNA), that may or may not have the same sequences,fixed to distinct active zones, one of the groups having been subjectedto the interaction of step b), and not the other.

According to a second “spatial” variant, the differential measurement ofstep c) is carried out with the measuring buffer on two groups of probemolecules arranged on distinct active zones, these two groups of probemolecules, that are for example of the same type, but may or may nothave the same sequences, having been subjected to different specificinteractions.

The probe molecules and/or the target biomolecules are, for example,DNA, RNA, PNA or protein molecules, or else small molecules such asvitamins. The reaction buffer and the measuring buffer are, for example,KCl. The salt concentration of the measuring buffer is, for example,greater than 0.002 mM and less than 20 mM, and it is, for example,greater than 0.005 mM and less than 20 mM, and in particular between0.005 mM and 15 mM, or else between 0.01 mM and 15 mM. The concentrationof the reaction buffer is, for example, between 20 mM and 1 M (molarconcentration).

The method according to the invention is compatible with a conventionaldetection of molecular interaction by fluorescence.

The method can be characterized in that said measurement of at least onepoint of the characteristic uses the application of a given voltage(U_(DS)) between the drain and the source of at least one transistor,and also the application, in a first case, of a given voltage (U_(GS))between the gate and the source of said transistor or, in a second case,of a given drain current (I_(D)), to said transistor. In the first case,the measurement of the characteristic point consists of the measurementof the drain current Id. In the second case, the voltage U_(GS) ismeasured.

According to one variant, at least one solution which constitutes areference or which contains target molecules is circulated through atleast one micro-fluidic channel so as to bring it into contact with atleast one active zone of a field-effect transistor.

It is, for example, possible to bring a reference solution (salt buffer,for example) and a solution containing target molecules successivelyinto contact with one or more active zones, or else (in parallel) withone or more different active zones. Two solutions of target moleculescan, for example, be brought into contact with one or more differentactive zones in parallel.

Other characteristics and advantages of the invention will emerge moreclearly on reading the description hereinafter, in conjunction with theattached drawings in which:

FIG. 1 represents two field-effect transistors of a detection chipcomprising a plurality of such transistors organized according to aone-dimensional or two-dimensional array of transistors;

FIG. 2 represents, viewed from above, details of a detection chip andthe arrangement of the active zones each corresponding to a field-effecttransistor;

FIG. 3 illustrates the electrical drain connections of the transmissionsof the one-dimensional or two-dimensional array;

FIG. 4 represents a device for depositing the solution onto selectedactive zones;

FIGS. 5 a to 5 c represent the results of experiments carried out undervarious experimental conditions;

FIGS. 6 a and 6 b show an electronic detection of DNA;

FIGS. 7 a and 7 b illustrate the use of the micro-fluidic channels;

and FIGS. 8 a to 8 h and 9 a to 9 d illustrate two examples of use ofthe invention.

FIGS. 1 to 3 illustrate a sensor having an array of field-effecttransistors FET on a silicon substrate. A transistor T₁ or T₂represented as a sectional view in FIG. 1 is provided with a sourceregion S and a drain region D which each present an electrical contactand which are surmounted by an insulating layer respectively 1 and 2,for example an SiO₂ thermal oxide. The active region 3 between thesources S and the drain D forms the gate region G of the transistor andhas a thin insulating layer 4, for example a layer of thermal SiO₂. Itis also possible not to have oxide on this active region. The activesurface is then delimited by a portion 4′ of the substrate which isstripped of insulating material.

Probe molecules, for example single-stranded DNA molecules, are fixed bya known method to at least some of the active surfaces 4 or 4′. For DNA,use is preferably made of depleted n-channel field-effect transistors(for which the charge carriers are electrons, which are more mobile,hence an increase in sensitivity) with a negative gate bias (i.e. theelectrolyte is negatively biased with respect to the semiconductor), theDNA being negatively charged (for an electrolyte of neutral pH).

The application of a source-drain voltage U_(SD) between the source Sand the drain D (U_(SD1) for T₁, and U_(SD2) for T₂) and of agate-source voltage U_(GS) between the electrolyte 6 and the source S(for example, by means of a single Ag/AgCl electrode E) induces atwo-dimensional gas of charge carriers at the Si/SiO₂ interface, or atthe Si/electrolyte interface of each transistor. A drain current I_(D)results therefrom, which current, for each transistor, dependssubstantially on the charge at the SiO₂/electrolyte or Si/electrolyteinterface. This interface which faces the channel between the source Sand the drain D is referred to as active surface.

The current I_(D) depends on the fixing of the probe molecules, forexample of the DNA molecules, to the active surface 4 or 4′.

It is possible to carry out a detection at constant source-gate voltageU_(SG) and at constant source-drain voltage U_(SD) by measuring thedrain current I_(Sd), or alternatively, as in examples 1 and 2hereinafter, at constant drain current I_(Sd) and at constantsource-drain voltage U_(SD) by measuring the source-gate voltage U_(SG).

As shown in FIGS. 2 and 3, n structures of field-effect transistor typeare integrated into a silicon substrate covered with an insulator (SiO₂or other) and provided with appropriate connections (metallization orpreferably doped conductive regions) by means of the electricalconnections of the source 10 and of the drain (D₁, . . . D_(n)) Unlike astandard MOS transistor structure, there is no metal gate electrode.This corresponds to the structure of ISFET (ion-sensitive field-effecttransistor) type. A substrate of SOI (silicon-on-insulator) type, whichprovides a higher sensitivity, is preferably used.

The various structures are laterally close to one another and theiractive surfaces are in contact with the same measuring solution. Atypical lateral dimension in current microelectronics is less than oneμm. In the DNA chip technology as used in the present invention, thelateral dimension is 5-10 μm for direct synthesis on the solid phase and50-100 μm in the case of fixing of the molecules using a dilution.

In the present parallel measurement configuration, several plots withvarious types of immobilized probe molecules are in contact with thesame solution, in particular measuring solution, and at least onetransistor structure is located below each plot. The use of severaltransistors per plot is possible in view of the abovementioneddimensions and permits redundancy in the detection.

An electrode E (Ag/AgCl, for example) is used to set the potential ofthe measuring solution 6 (electrolyte) with respect to the siliconstructure that it covers and to set the operating point of the sensors(transistors). The potential of the electrolyte 6 can, in certain cases,be equal to zero. The measuring solution 6 which bathes the sensorscontains ions at a concentration which gives sufficient conductivity andwhich does not give rise to too great a screening of the activesurfaces. It preferably has a neutral pH.

The technique proposed makes it possible to facilitate the detectionusing various approaches.

A—Characterizing the Layers by Means of Electron Measurements

Electron measurement allows a rough characterization of the(electrostatic) states of the molecular layers deposited on thetransistors. Electron measurements are carried out between the varioussurface preparation steps. Each chemical treatment step or moleculedepositing step induces a shift in the voltage measured and thedifferences between the transistors reflect any possible nonhomogeneity.All these shifts then provide a characterization of the system withmultiple deposited layers.

In other words, it is thus possible to verify the “electrochemical”state of the array of sensors. It is possible to compare with referencevalues measured for conditions that had been optimized beforehand andthe aging of the structures can be monitored (for example, thedegradation of the arrays by charge deposits and other effects on theSiO₂ oxide). In addition, comparison of two electron measurementsseparated by a small period of time (and, optionally, intermediaterinsing) makes it possible to verify the absence of any substantialdrift in the electronic signal. These points are important since theresponse of the FET field-effect transistors to the immobilization ofmolecules on the active surface depends on all the layers on the activesurface. The procedure helps to reproducibly obtain a suitablesensitivity (suitable for the amount of molecules that it is desired todetect) and a sufficient specificity.

Once the sensor array is known, the measurements can be repeated withoutperforming further verifications.

B—Separating the Recognition Reaction from the Detection Step

The depositing of the probes, the recognition reaction and the electronmeasurement can be carried out in various buffers. This makes itpossible to optimize these steps largely independently. The example of ahybridization with a salt concentration of 50 mM coupled to anelectronic detection with a salt concentration of 0.01 mM is inparticular shown hereinafter.

C—Differential Measurement

The differential approach, which is the subject of applicationPCT/FR02/04283, of which the present application claims the priority, isa very important element here. Compared with characterization of thelayers by electron measurement, it makes it possible to detectnonhomogeneities. With respect to the separation of the recognitionreaction from the detection step, it even appears to be essential, sincethe shift in the value of the voltage U_(GS) induced by a change inbuffer often shows a variation that is greater than the signalcorresponding to the specific interaction that it is desired to detect.

D—Active Structures

The use of the FET field-effect structures permits a characteristic sizeof the order of 1 micron. The approach by alternating current ACmeasurement is used by Fritz et al. (Proceedings Nat. Acad. Sci., USA,Vol. 99, p. 14142 (2002)). The alternative approach of an SiO₂/Sipassive structure with neither drain nor source (referred to as“impedance spectroscopy”) requires a surface area of approximately 50microns by 50 microns because of the parasitic capacity due to theconnections [(see Wiegand et al., Review of Scientific Instruments No.71, 2309 (2000)]. This difficulty could be bypassed through a treatmentof the alternating signal in the actual chip, in the vicinity of thesensor.

Even in this case, the measurement electronics would be more complicatedthan those used in the context of the present invention for the directcurrent DC measurement based on the FET field-effect transistors. Theactive structures (FET) are therefore more suitable for miniaturization.By using active surface areas of 2 microns by 20 microns, the noise ofthe FET transistors is not limiting.

The method for detecting molecular recognitions is based on an approachby comparison, in particular differential comparison. The measurementis, for example, carried out using several transistor structures. Themeasurement may be differential with respect to the various types ofmolecules grafted and may optionally include several transistors pertype of molecule. It makes it possible to compare signals before/afterthe interaction reaction which reveals the molecular recognition (and/orthe evolution during this reaction). It will be noted that the referencemeasurement can be carried out in the measuring buffer having the secondconcentration, but also in another buffer, for example in said reactionbuffer having a first salt concentration.

The method according to the invention makes it possible to circumventthe difficulties associated with the sensitivity of an individual sensorto the pH and to the ionic strengths and those associated with avariability from one individual transistor to the other (this includesthe transistor structure and the quality of the fixing of the probes).

A method according to one embodiment uses the following steps:

a) homogeneous treatments of the entire insulating surface in order toprepare the fixing of the probe molecules;

b) local grafting of various types of probe molecules onto at least someof the individual active surfaces;

c) optionally, homogeneous rinsing;

d) electron measurements: the measuring electrolyte is added, theelectrode is immersed and the transistors are measured (for example, oneor more points of the characteristic I_(D) as a function of U_(SD) andof U_(SG)), and the results obtained are compared according to thetransistors;e) optionally, homogeneous rinsing;f) addition of the solution of target molecules in the presence ofelectrolyte and recognition reaction;g) optionally, homogeneous rinsing;h) electron measurement, as (d).

Some transistors which have not been brought into contact with probemolecules (or else a single transistor) can serve as controls. Theircharacteristics are measured after addition of the measuring electrolytewhich, for example, bathes all the transistors.

The grafting of the probe molecules is carried out by depositingmicrodroplets approximately 100 μm in diameter onto the active surfacesof the transistors using metal micro-pins which are commerciallyavailable, or else a commercial microdeposition system (for example,nanoplotter NP1 from the company Ge Sim).

As shown in FIG. 3, the array of n transistors (for example, n=96transistors) has n drain connections D₁, D₂ . . . D_(n) and 2connections (not represented) equivalent to the common source. Theseries resistances R_(c) associated with these connections have valueswhich depend on the index 1 . . . n of the drain.

The values of these resistances R_(c) produced, for example, by silicondoping, are not negligible, but they can be corrected.

To this effect, the drain connection resistances R_(c) are, for example,calculated from the geometric links and cross sections of the dopedlines, the resistivity of which is known. The calculation is comparedwith a measurement of the resistance as a function of the drain index byapplying a DC voltage (for example, U_(SD)=0.1 V and U_(SG)=2 V).

An installation such as that represented in FIG. 4 can be used toimplement the method: a platform 12 is placed on a table 10, saidplatform incorporating a control device comprising a microcontroller fora table 11 providing movement in three perpendicular directions X, Y andZ. A chip 15 incorporating the array of n transistors is placed on asupport 14. Another platform 20 comprising a table 21 providing movementin the three directions X, Y and Z is used to move an arm 22 carrying amicro-pin or a pipette 23 for depositing the microdroplets onto at leastsome of the n transistors. An objective 17 and/or a camera coupled to ascreen 19 make it possible to observe the deposition of themicrodroplets and to control the operations.

Drain current I_(D) measurements are carried out with, for example,U_(SG)=1 V and U_(SD)=0.9 V and a deposited electrolyte of neutral pHwhich consists of KCl at a content of 0.1 millimole per liter. Since thetransistors (p-channel storage transistors) have their sourcesinterconnected, the source voltage or the gate voltage can serve asvoltage reference (for example, the mass voltage).

Before these measurements, an overall treatment of the surface of theSi/SiO₂ structure is performed by incubation for 1-2 minutes insulfochromic acid and rinsing under a stream of deionized water and thenincubation for 3 to 5 minutes in a solution of NaOH (60 μl 16N NaOH, 420μl of ethanol and 220 μl of water) and, finally, rinsing under a streamof deionized water.

The difference between two measurements carried out before localdeposition but before and after rinsing with water is shown as smallsquares in FIG. 5 a. The crosses represent the difference between ameasurement carried out after local deposition of two differentsolutions and a measurement carried out before deposition (themeasurement carried out before the rinsing with water).

Using a commercial pin 23 (Telechem SMP3B) mounted on the device 22shown in FIG. 4, a solution of poly-L-lysine is deposited onto thetransistors 64-69, the transistors 74-79 and the transistors 87-91.

Solution: poly-L-lysine (0.01% weight/volume “w/v” final concentration(P8920, Sigma)) in a 0.1×PBS buffer at pH 7.

After the local depositions, the sample is dried for 15 minutes in ahumid atmosphere and then for 5 minutes at 50° C.

The poly-L-lysine is positive in the measuring electrolyte (neutral pH)due to the ionized amine groups. The decrease in current observed on thepoly-L-lysine deposits is compatible with the adsorption of a positivecharge onto the surface.

The difference in surface potential ΔU_(SG) corresponding to themeasurement before/after deposition is measured. In order to determineΔU_(SG), the two-dimensional characteristic, for example I_(D)(U_(SG),U_(SD)), is measured and the intrinsic characteristics of the 96transistors are determined by numerically correcting the characteristicsmeasured as a function of the resistances R_(c) of the drain lines inseries. The modification of the condition of the SiO₂ interface inducesa change in the intrinsic characteristic which corresponds to a shiftΔU_(SG) at constant U_(SD) and constant drain current I_(D). This shiftmakes it possible to directly obtain an independent measurement of theoperating point of the transistor, unlike the change in current ΔI_(D)presented in FIG. 5 a. The value ΔU_(SG) makes it possible, in firstapproximation, to quantify the change in the SiO₂/liquid interfaceinduced by the local deposit. According to a variant, U_(SG) is variedso as to keep I_(D) constant.

FIGS. 5 a to 5 c show differential measurements carried out before andafter deposition of poly-L-lysine (FIG. 5 a), carried out as a functionof the concentration of KCl (FIG. 5 b), and carried out as a function ofthe concentration of deposited poly-L-lysine.

In FIG. 5 a, the variations ΔI_(D) in the drain current I_(D) arerepresented on the y-axis for each of the transistors 60 to 96identified on the x-axis (U_(SG)=1 V, U_(SD)=0.9 V and electrolyte KClat 0.1 mM). The differences ΔI_(D) between two measurements carried outbefore a local deposition but separated by rinsing with water arerepresented by circles. The differences ΔI_(D) corresponding tomeasurements carried out before and after a local deposition ofpoly-L-lysine are represented by stars. After the local deposition, thesample is left at ambient temperature for 15 minutes in humid medium,before being dried at 50° C. for 5 minutes. The dilution C_(o) of thepoly-L-lysine is 0.01% weight/volume “W/V” final concentration (P8920,Sigma) in 0.1×PBS buffer at pH 7.

In FIG. 5 b, the differences ΔU_(SG) in the source-gate voltage U_(SG)are measured on some of the transistors of an array of 62 FETtransistors with U_(SD)=1.2 V and I_(D)=50 μA. The differences between areference measurement (carried out before local deposition and with aconcentration of KCl of 0.01 mM) and two series of measurements (carriedout after local deposition of poly-L-lysine and with variousconcentrations of KCl) are represented by circles and stars. Here, alocal deposition of poly-L-lysine was carried out in two distinct zoneswith the same dilution C_(o) as in the case of FIG. 5 a. In each of thetwo series of measurement, the concentration of KCl in the measuringbuffer is varied between 0.01 mM and 100 mM, the range including thevalues 0.1 mM, 1 mM and 10 mM. The surface is rinsed with water betweenthe two series of measurement. A notable sensitivity of the detection ofpoly-L-lysine is observed for KCl concentrations of between 0.01 mM and1 mM, and the height of the peaks gradually decreases beyond thesevalues.

FIG. 5 c shows the variations ΔU_(SG) of the voltage U_(SG) as afunction of the concentration of polymer deposited (poly-L-lysine), i.e.2C_(o), C_(o), C_(o)/2, C_(o)/4, C_(o)/8, in a 0.1×PBS buffer, pH 7,C_(o) having the value indicated for the measurements in FIG. 5 a. Themeasuring conditions are as follows: U_(SD)=1 V, I_(D)=100 μA, and aconcentration of 0.01 mM for KCl. These measurements show that there isno advantage, under the experimental conditions chosen, in increasingthe concentration beyond C_(o).

FIGS. 6 a and 6 b show the electronic detection of DNA. The voltagesU_(SG) and the variations ΔU_(SG) in the voltage U_(SG) correspond to anoperating point U_(SD)=1 V, I_(D)=100 μA, and a KCl concentration of0.01 mM. They are obtained from the characteristic I_(D)(U_(SG), U_(SD))and are recorded on the curves with the FET transistor number (1 to 96)on the x-axis.

The stars represent the measurement after initial surface treatment withsodium hydroxide. The circles represent the measurement after incubationof poly-L-lysine on the entire array. In order to allow immobilizationof DNA, the array of FET transistors is incubated for 30 minutes in adilution of poly-L-lysine (concentration Co). Next, without any priordrying, rinsing is carried out with water, followed by air-drying. Theincubation results in shifts in the voltage U_(SG) by a value of 97±50mV (statistical value over 67 surfaces prepared), which reduce thevariations between transistors in the electronic signal. These shiftsare compatible with those observed with the values measured in relationto FIG. 5 c on local deposits at the same concentration. The squaresrepresent the measurements after local deposition of oligonucleotides(5′ Cy-5 modified 20-mer, concentration 50 μM in deionized water) aroundthe transistors Nos. 30, 60 and 90. The microfluorescence image of theabovementioned three DNA points is represented in level of gray andabove FIG. 6 a.

FIG. 6 b shows the electronic detection and detection by fluorescence ofCy5 modified oligonucleotides. The points represented by stars wereobtained by the difference ΔU_(SG) between two electron measurementscarried out before and after 4 local depositions with differentconcentrations of DNA (ref.=0 μM, 5 μM, 10 μM, 20 μM). They show thevariation ΔU_(SG) in the voltage U_(SG) which is observed in thecharacteristics of the transistors and which is due to the localdeposits of DNA. The squares show the intensity of the fluorescencemeasured on the dried FET transistors, once the electron measurement hasbeen carried out with the electrolyte. It will be noted that the sameelectronic detection is obtained with oligonucleotides of the same type,but which are not modified.

FIGS. 7 a and 7 b show an integrated circuit having transistors Tarranged along a line (or several lines). Two microfluidic channels (forexample parallel) C₁ and C₂ of a substrate 30 make it possible to bringone or more field-effect transistors T into contact with a referencesolution or a solution containing target molecules which circulate in achannel C₁ and/or C₂. The material of a substrate 30 which comprises themicrofluidic channels (or capillaries) can be a PDMS(polydimethylsiloxane) polymer or the like, a glass, silicon, etc.

It is thus possible to carry out differential measurements using twosolutions which circulate in the two channels C₁ and C₂. It is alsopossible to prepare a large number of such microfluidic channels on thesame substrate 30, the substrate in which they are arranged beinginterlinked with the semi-conductor substrate into which the FETfield-effect transistors are integrated. It is also possible to measurea variation inside a given channel. This variation may be over time. Itis also possible to inject various solutions into a capillary, and theconcentration profile remains unchanged along the channel, even far fromthe point of injection. Reference will be made to the article by Paul J.A. Kenis et al., entitled “Microfabrication inside capillaries usingmultiphase laminar flow patterning”, published in Science, vol. 285,Jul. 2, 1999, pp. 83-85 (in particular FIG. 1 a).

An analytical technique using microfluidics is described in the article“Monolithic integrated microfluidic DNA amplification and capillaryelectrophoresis analysis system” by Eric T. Lagally et al., published inSensors and Actuators B 63 (2000), pp. 138-146.

The detection of at least one specific interaction between probebiomolecules and the target biomolecules is advantageously carried outby using a measuring buffer for which the salt concentration (forexample, KCl) is lower than that of the reaction buffer.

The biomolecules concerned (sources or targets) can, for example, beDNA, RNA, proteins and vitamins.

The specific interactions can, for example, be DNA-DNA, DNA-RNA,DNA-protein, RNA-protein, protein-protein or else vitamin-proteininteractions. The DNA can be chemically synthesized oligonucleotide DNA.Inter-actions can also be carried out with a peptide nucleic acid “PNA”.

Additional measuring steps have been added in order to verify thesurface condition of the sensor (FET transistors), the quality of thefixing of the probe biomolecules and the reproducibility of themeasurements.

EXAMPLES Products

NaOH: 60 microliters of 16N NaOH, 420 microliters of ethanol, 220microliters of water. PLL: solution of poly-L-lysine, P8920 (Sigma),0.01% w/v in a 0.1×PBS buffer. Oligonucleotide ARS3: 5′ CCG CGA ACT GACTCT CCG CC (SEQ ID NO: 1); Oligonucleotide ARS5: 5′ CAG GCG CGA GGG CTGACG TT (SEQ ID NO: 2); Oligonucleotide Cy3-ARS3sense: (complementary toARS3 and with Cy3 fluorophore) Oligonucleotide Cy5-ARS5sense:(complementary to ARS5 and with Cy5 fluorophore).

Example 1 Hybridization in a 50 mM KCl Buffer and Measurement with a0.01 mM KCl Buffer

The example shows that it is possible to obtain higher signals bydecreasing the salt concentration of the measuring electrolyte comparedwith that of the hybridization. The specific hybridization is verifiedby means of a control measurement by fluorescence.

1. Overall Treatment of the SiO₂ Surface

Incubation for 1 minute in sulfochromic acid, and then rinsing in astream of deionized water, and drying with compressed air. This cycle ofincubation, rinsing, drying is repeated once.

Incubation for 4 minutes in the NaOH solution.

Rinsing with water and drying.

2. Electron Measurement after NaOH Treatment

Measuring buffer: KCl at a concentration of 0.01 mM (see FIG. 8 a).

This measurement is followed by rinsing with water and drying.

3. Overall Poly-L-Lysine Treatment

Incubation is carried out for 2 h.

This incubation is followed by rinsing with water and drying.

4. Electron Measurement “PL1”

Measuring buffer: 0.01 mM KCl (see FIG. 8 a).

This measurement is followed by rinsing with water and drying.

5. Electron Measurement “PL2”

Measuring buffer: 0.01 mM KCl.

This measurement is followed by rinsing with water and drying.

The aim of this second measurement is to verify the stability of themeasurement at this stage.

6. DNA Probe Deposition

0.2 Microliters of a solution containing the oligonucleotide Ars5 isdeposited onto the left portion of the FET array, with a micropipette.0.2 Microliters of a solution containing the oligonucleotide Ars3 isdeposited onto the right portion of the array. In the two cases, thedilutions contain 1 micromole of oligonucleotide in a 50 mM KCl buffer.Incubation is carried out for 15 minutes, in a humid atmosphere so as toprevent drying.

This incubation is followed by rinsing with water and drying.

7. Electron Measurement “Probe 1”

A measuring buffer, 0.01 mM KCl, is used.

FIG. 8 b shows the differences delta U_(GS) between the “probe 1”measurement carried out after deposition of the probe biomolecules andthe PL2 measurement (step 5) carried out before this deposition.

The “probe 1-PL2” curve (KCl concentration: 0.01 mM) shows a shift of 25mV for the two regions of deposition on the left (deposition of Ars3 onthe transistors 1 to 13) and on the right (deposition of Ars5 on thetransistors 21 to 31). This value means that there is a sufficientconcentration of probe biomolecules, that is not yet too close tosaturation.

Pumping of the electrolyte is then performed, and said electrolyte isreplaced with 1 ml of 50 mM KCl, without drying.

8. Electron Measurement “Probe 2”

Measuring buffer: KCl at a concentration of 50 mM.

As shown in FIG. 8 b, the deposits are no longer seen.

No rinsing. An electron measurement “probe 3” is carried out (50 mM KCl)in order to verify the stability (FIG. 8 c).

9. Hybridization 1 (with Cy5-Ars5Sense)

During the “probe 2” measurement (step 8), there was 1 ml of KCl on theFET transistors.

Next, 100 μl of Cy5Ars5sense are added directly (without rinsing).

The final dilution of oligonucleotides is then of the order of 100 nM.Agitation is carried out by pumping and this solution is redepositedtwice. After agitation, the recognition reaction takes place for 5minutes in the dark (so as to prevent bleaching of the fluorophores).

After 5 minutes, rinsing is performed: pumping of the electrolyte andaddition of 1 ml of 50 mM KCl, agitation, followed by further pumping,etc. This cycle is repeated 3 times. These rinses stop the reaction. Atno time during this step is there any drying on the surface.

10. Electron Measurement “Hyb1”

The electrode is immersed in the 50 mM KCl buffer and the electronmeasurement is carried out.

The resulting Hyb1-probe 3 curve is represented in FIG. 8 d.

The shift delta U_(SG) between the measurements referred to as Hyb1 andprobe 3 is approximately +3.9 mV over the Ars3 region and approximately+2.3 mV over the Ars5 region (here, the probe biomolecules and thetarget biomolecules have complementary sequences). This shift isapproximately −1.6 mV over the central region that has no DNA probedeposition (transistors 14 to 20).

After this measurement, rinsing is performed: pumping of the electrolyteand addition of 1 ml of 0.01 mM KCl, agitation, repumping, etc, repeated5 times. At no time during this step is there any drying on the surface.

11. Electron Measurement “Hyb12”

The measuring electrode is immersed in the 0.01 mM KCl buffer and theelectron measurement is carried out.

The resulting curve (Hyb12-probe 1) is represented in FIG. 8 e. Theshift signs correspond to those which were observed at 50 mM (“Hyb1”measurement), but the levels of these shifts are greater.

In the Ars5 (Ars3) region, a mean shift of +40 mV (+49 mV) is observedbetween the “Hyb12” and “probe 1” measurements, carried out at 0.01 mMKCl.

12. Hybridization 2 (with Cy3-Ars3sense)

The 0.01 mM KCl electrolyte is pumped and is replaced with 1 ml of 50 mMKCl. Next, 100 μl of 1 μM Cy3Ars3sense are added directly (withoutrinsing).

The final dilution of oligonucleotides is then of the order of 100 nM.

Agitation is carried out by pumping and this solution is redepositedtwice. After agitation, the recognition reaction takes place for 5minutes in the dark.

Once these 5 minutes have elapsed, rinsing is performed, followed bypumping of the electrolyte, after which 1 ml of 50 mM KCl is added.Agitation and pumping are again performed. This cycle (rinsing,agitation, pumping) is repeated 3 times. These rinses terminate thereaction. At no time during this step is there any drying on thesurface.

13. Electron Measurement “Hyb2”

The electrode is immersed in the 50 mM KCl buffer and the electronmeasurement is carried out. The result of these measurements isrepresented by the Hyb2-probe 3 curve in FIG. 8 d.

In FIG. 8 d, the difference delta U_(SG) between the abovementionedmeasurements Hyb1 and probe 3 is observed to be more positive on theArs3 region (on the left) than on the Ars5 region (on the right, wherethe probes and targets have complementary sequences for hybridization1). This tendency is reversed for the difference between Hyb2 and probe2, in accordance with the fact that, for hybridization 2, the probe andtarget biomolecules have complementary sequences on the Ars3 region.

After this measurement, rinsing is performed: pumping of the electrolyteand addition of 1 ml of 0.01 mM KCl, agitation, repumping, etc, repeated5 times.

At no time during this step is there any drying on the surface.

14. Electron Measurement “Hyb22”

The electrode is immersed in the 0.01 mM KCl buffer and the electronmeasurement is carried out. The result is represented by the Hyb22-probe1 curve in FIG. 8 f.

The shift signs are reversed with respect to those observed at 50 mM(“Hyb2” measurement) and the signals are higher.

In the Ars5 (Ars3) region, a mean shift of −11 mV (+1 mV) is observedbetween the “Hyb22” and “Hyb12” measurements.

After this measurement, rinsing with water and drying are carried out.

15. Fluorescence Measurements

The specific hybridization is verified with a two-color fluorescencemeasuring device.

With excitation at 633 nm (Cy5), more fluorescence is observed on theArs5 region FIG. 8 h).

With excitation at 532 nm (Cy3), more fluorescence is observed on theArs3 region (FIG. 8 g).

Conclusion

Measurements at 50 mM and hybridization with Ars5Sense: the shift of theArs5 region is 1.6 mV more negative than that of the Ars3 region.

Measurements at 50 mM and hybridization with Ars3Sense: the shift of theArs3 region is 5.4 mV more negative than that of the Ars5 region.

Measurements at 0.01 mM and hybridization with Ars5Sense: the shift ofthe Ars5 region is 9 mV more negative than that of the Ars3 region.

Measurements at 0.01 mM and hybridization with Ars5Sense: the shift ofthe Ars3 region is 22 mV more negative than that of the Ars3 region.

The specific hybridization is verified by the fluorescence measurement.

Example 2 Hybridization with Microdeposition and 20 mM/0.01 mM BufferChange

Steps 1 to 5 are identical to those in example 1.

6. DNA Probe Deposition

Using a commercial microdeposition system (GeSim, Rossendorf, Germany,Nano-plotterNP1), substantially 0.2 nl of a solution containing theoligonucleotide Ars5 is deposited on the left portion of the FETtransistor array. 0.2 nl of a solution containing the oligonucleotideArs3 is deposited on the right portion of this array. A mixture of Ars5and of Ars3 is deposited at the center. In all cases, the dilutionscontain 1 μm (1 micromole) of oligonucleotide in a buffer of deionizedwater. The probes dry a few seconds after the deposition at normaltemperature and humidity.

No rinsing.

7. Electron Measurement “Probe 1”

Measuring buffer: 0.01 mM KCl.

The probe 1-PL2 curve in FIG. 9 a shows the differences delta U_(SG)between measurements carried out after the probe deposition and the PL2measurement carried out before this deposition (with the same measuringbuffer).

The “probe 1-PL2” curve shows that the deposition of theoligonucleotides induced shifts delta U_(SG) of between −20 and −25 mV.

The measurement is followed by pumping of the electrolyte andreplacement thereof with 1 ml of 20 mM KCl, without drying.

8. Electron Measurement “Probe 2”

Measuring buffer: 20 mM KCl.

The results are represented in FIG. 9 a by the probe 1-PL2 curve (20 mMmeasuring buffer). The deposits are still seen, but the peaks aresmaller than with the 0.01 mM measuring buffer.

No rinsing.

9. Electron Measurements “Probe 3” and “Probe 4”

Measuring buffer: 20 mM KCl.

The aim of these measurements is to verify the stability.

No rinsing.

10. Hybridization 1 (with Cy3-Ars5Sense)

During the “probe 4” measurement, there was 1 ml of KCl on the FETs.Next, 100 μl of 1 μM Cy3Ars5Sense are added directly (without rinsing).The final dilution of oligonucleotides is then approximately 100 nM.Agitation is carried out by pumping and this solution is redepositedtwice. After agitation, the recognition reaction takes place for 5minutes in the dark.

After these 5 minutes, rinsing is performed: pumping of the electrolyteand addition of 1 ml of 20 mM KCl, this cycle being repeated 3 times.These rinses terminate the reaction. At no time during this step isthere any drying on the surface.

11. Electron Measurement “Hyb1”

The electrode is immersed in the 20 mM KCl buffer and the electronmeasurement is carried out.

The result is represented by the “Hyb1-probe 4” curve in FIG. 9 b.

The shift delta U_(SG) between the abovementioned Hyb1 and probe 4measurements is approximately −21 mV over the Ars3 region (transistors71 to 81) and approximately −25 mV over the Ars5 region (transistors 11to 19). Here, on the Ars5 region, the probe and target biomolecules havecomplementary sequences.

After this measurement, rinsing is performed: pumping of the electrolyteand addition of 1 ml of 0.01 mM KCl, agitation, repumping, etc, repeated5 times.

At no time during this step is there any drying on the surface.

12. Electron Measurement “Hyb12”

The electrode is immersed in the 0.01 mM KCl buffer and the electronmeasurement is carried out. The result is represented in FIG. 9 c.

The shift signs correspond to those which were observed with a measuringbuffer having a concentration of 20 mM, but the signals are higher.

In the Ars5 (Ars3) region, a mean shift of −61 mV (−41 mV) is observedbetween the “Hyb12” and “probe 1” measurements.

After this measurement, rinsing is performed: pumping of the electrolyteand addition of 1 ml of 20 mM KCl, agitation, repumping, etc, repeated 5times. At no time during this step is there any drying on the surface.

13. Fluorescence Measurement

The specific hybridization is verified using a two-color fluorescencemeasuring device.

With excitation of 532 nm (FIG. 9 d), more fluorescence is observed onthe Ars5 region. This confirms the specific hybridization.

Conclusions

Measurements at 20 mM and hybridization with Ars5Sense: the shift of theArs5 region is 3.8 mV more negative than that of the Ars3 region.

Measurements at 0.01 mM and hybridization with Ars5Sense: the shift ofthe Ars5 region is 20.7 mV more negative than that of the Ars3 region.

The specific hybridization is verified by the fluorescence measurement.

The reference measurement carried out with probe molecules that have notbeen subjected or not yet been subjected to an interaction with targetmolecules can be performed on one or more active zones distinct fromthat or those where a said interaction reaction with target moleculestakes place. The active zones of the same integrated circuit can be usedfor various interactions, which makes it possible to perform, inparallel, several measurements of different types corresponding, forexample, to various interactions, and/or to the study of the varioustarget biomolecules. It is thus possible to detect several interactionswith respect to one or more reference measurements carried out on probemolecules not subjected to interactions. It is also possible to detectinteractions by means of comparisons between one another of measurementscarried out on molecules that have been subjected to variousinteractions, for example between probe molecules of the same type, forexample DNA, that may or may not have the same sequences, and identicalor different target biomolecules.

Two examples of potential applications of the invention are givenhereinafter:

One example is the study of gene expression. Interest here centers onthe relative amount of the various messenger RNA molecules extractedfrom cells. DNA probe molecules which carry different base sequences aregrafted at different sites of the surface of the chip. Each DNA probe ischosen so as to interact specifically with one type of RNA molecule(characterized by its sequence). The relative number of RNA moleculeshybridized on two regions of different probes gives a relative abundanceof two types of RNA molecules (and with this, the relative level ofexpression of the corresponding two genes). The advantage is that alarge number of genes can be followed in parallel, for developmentalstudies, for genetic characterization of pathologies, for molecularanalysis of the effect of medicinal products, etc.

Another example concerns the detection of mutations on DNA chips. Here,the intention is to analyze the DNA of a patient by investigating, inparallel, the presence (or absence) of several known mutations. Forthis, various oligonucleotides (typically 12-mers) are grafted atvarious sites of the chip. The base sequences and the hybridizationconditions (salt, temperature, etc) are optimized so that the presenceof a mutation (even a point mutation) induces a measurable difference inthe degree of hybridization of the various probes. The target moleculesample often consists here of double-stranded DNA molecules obtained by“PCR” from a small amount of genomic DNA from the patient.

In the two cases, interest is centered on the difference in the signalsinduced by the hybridization of a complex sample containing a largenumber of different target biomolecules, on a chip with several regionsof different probes (often the same type of molecule but with adifferent sequence). This configuration is very advantageous forelectronically detecting interactions.

It is understood that the expressions “two different interactions” and“other interaction” cover the situation where this difference ininteraction corresponds to two probes of different type or sequence,that are in contact with the same target molecule solution.

The detection of specific interactivity can be carried out on one ormore gate regions of the FET field-effect transistor array. Theadvantage of using several gate regions makes it possible to demonstratethe nonhomogeneities.

Alternatively, the use of a single transistor of the array is possiblefor probe biomolecules of a given type and a given interaction. If atransistor with large dimensions is used, an averaged measurement of thenonhomogeneities is directly obtained.

1. A method for electronically detecting at least one specificinteraction between probe molecules fixed to at least one active zone ofa sensor and target biomolecules, wherein said sensor comprises an arrayof field-effect transistors, each of which has a source region, a drainregion, and a gate region which constitutes an active zone on which saidspecific interaction is to be detected, and wherein said methodcomprises: a) contacting at least one active zone with a probe moleculeof a given type fixed to said active zone, b) contacting at least someof the probe molecules with target biomolecules capable of interactingwith said probe molecules in a reaction buffer having a first saltconcentration, c) measuring at least one point of a drain current, asource-gate voltage, or a source-drain voltage characteristic of atleast one transistor of said array to detect said specific interactionat least for a measurement point obtained in a measuring buffer having asecond salt concentration that is lower than the first concentration forprobe molecules having been subjected to said specific interaction, saidmeasurement being conducted spatially by means of a difference betweensaid measurement point and a reference point, in said measuring buffer,for two groups of probe molecules fixed to distinct active zones, themeasuring point being obtained for probe molecules having been subjectedto the interaction of step b) and the reference point being obtained forprobe molecules not having been subjected to the interaction of step b).2. The method of claim 1, wherein said reference point is determinedfrom probe molecules of the same type as those that were subjected tosaid specific interaction, and having even the same sequence or adifferent sequence.
 3. The method of claim 1, wherein said the probemolecules subjected to said two different interactions are of the sametype, whether or not they have identical sequences.
 4. The method ofclaim 1, wherein said measuring at least one point of a drain current, asource-gate voltage, or a source-drain voltage characteristic comprisesapplying a given voltage (U_(DS)) between the drain and the source of atleast one transistor, and applying, in a first case, a given voltage(U_(GS)) between the gate and the source of said transistor or, in asecond case, a given drain current (I_(D)), to said transistor.
 5. Themethod of claim 4, wherein, in the first case, the point is obtained bymeasuring the drain current I_(D) and, in the second case, by measuringthe voltage U_(GS) between the gate and the source.
 6. The method ofclaim 1, wherein the measuring buffer is KCl.
 7. The method of claim 1,wherein the concentration of the reaction buffer is between 20 mM and 1M.
 8. The method of claim 7, wherein the concentration of the measuringbuffer is greater than 0.002 mM and less than 20 mM.
 9. The method ofclaim 8, wherein the concentration of the measuring buffer is at leastequal to 0.01 mM.
 10. The method of claim 8, wherein the concentrationof the measuring buffer is at most equal to 15 mM.
 11. The method ofclaim 1, wherein the passage between one buffer and a buffer of lowerconcentration is separated by a rinsing step.
 12. The method of claim 1,wherein the probe molecules are molecules, in particular biomolecules,capable of being recognized by a type of target biomolecule.
 13. Themethod of claim 12, wherein the probe molecules and/or the targetbiomolecules are DNA, RNA or protein molecules, or else vitamins. 14.The method of claim 13, wherein the probe biomolecules are DNA moleculesand in that the field-effect transistors are of the depleted n-channeltype, with a negative gate bias.
 15. The method of claim 1, wherein saidmethod further comprises, before a), at least one control measurementstep with a said measuring buffer.
 16. The method of claim 1, whereinsaid method further comprises the circulation of at least one solutionwhich constitutes a reference or which contains target molecules throughat least one microfluidic channel so as to bring it into contact with atleast one said field-effect transistor.
 17. The method of claim 9,wherein the concentration of the measuring buffer is at most equal to 15mM.